BackgroundSurgical treatment options for horses with overriding dorsal spinous processes include interspinous ligament desmotomy and partial spinous process ostectomy. The impact of spinal surgery on the three‐dimensional biomechanics of the equine thoracolumbar spine and the epaxial musculature is unclear.ObjectivesTo investigate the influence of interspinous ligament desmotomy and cranial wedge ostectomy on the biomechanics of the equine thoracolumbar spine and the paraspinal Musculi multifidi.Study designEx‐vivo experiments.MethodsTwelve equine thoracolumbar spine specimens were mounted in a custom‐made mechanical test rig. Based on computed tomographic imaging, distances between dorsal spinous processes and the spinal range of motion (lateral bending, axial rotation, flexion, extension) were compared before and after desmotomy and cranial wedge ostectomy performed at two or five surgical sites. Anatomical dissection was subsequently conducted to document surgical trauma to the Musculi multifidi following desmotomy.ResultsThe distance between spinous processes in neutral position did not increase significantly after desmotomy (median preoperative = 7.2 mm, interquartile range [IQR] = 3.6 mm; median postoperative = 7.4 mm, IQR = 3.7 mm; p = 0.09), but increased significantly after ostectomy (median preoperative = 8.8 mm, IQR = 4.2 mm; median postoperative = 13 mm, IQR = 6.1 mm; p < 0.001). Both surgical procedures significantly increased the rotational spinal range of motion (p = 0.001), particularly at the level T14/T15 (median preoperative = 6.4°, IQR = 3.2°; median postoperative = 8.2°, IQR = 3.5°; increase = 28.1%; p = 0.02). Musculi multifidi injury was evident at all desmotomy sites.Main limitationsEx‐vivo study with limited sample size.ConclusionsNeither interspinous ligament desmotomy nor cranial wedge ostectomy resulted in an increased range of motion during flexion, extension or lateral bending but both procedures influenced the rotational component of the equine thoracolumbar spinal mobility.